1. Field of the Invention
The invention relates to procedures and devices for fragmenting molecular ions, preferably biomolecular ions, in high-frequency quadrupole ion trap mass spectrometers.
2. Description of the Related Art
Paul ion traps consist of a ring electrode and two end cap electrodes, whereby a storage RF voltage is usually fed to the ring electrode; however, other modes of operation can also be implemented. Ions can be stored in the interior of the ion trap within the quadrupolar RF field. The ion traps can be used as mass spectrometers in which the stored ions are ejected mass specifically and measured by a secondary electron multiplier. Several different methods are known for ion ejection which will not be discussed any further here.
The RF voltage at the ring electrode is very high, and in customary ion trap mass spectrometers it can be ramped up, during a mass scan, to maximum voltages between 15 and 30 kV (peak to peak). The frequency is in the range of 1 MHz. In the interior a mainly quadrupolar field is generated which oscillates with the RF voltage and drives the ions above a certain mass threshold back into the center, which results in so-called secondary oscillations of the ions in the trap. The retroactive force in the ion trap is sometimes described as a so-called pseudopotential, which is determined by the average of the forces of the real potential over time. At the center there is a saddle point for the oscillating real potential which quadratically falls off from the saddle point to the ring electrode, and quadratically increases from the saddle point to the end cap electrode (or vice versa, depending on the phase of the RF voltage).
Ion trap mass spectrometers possess properties which make their use interesting for many types of analysis. For example, selected ion types (so called xe2x80x9cparent ionsxe2x80x9d) can be isolated and fragmented in the ion trap. The spectra arising from such fragment ions are termed xe2x80x9cfragment ion spectraxe2x80x9d or xe2x80x9cdaughter ion spectraxe2x80x9d of the corresponding parent ions. xe2x80x9cGranddaughter spectraxe2x80x9d as fragment ion spectra of selected daughter ions can also be measured. Until now, ions have usually been fragmented by a large number of collisions with a collision gas; the oscillation of the ions to be fragmented is excited by a bipolar alternating field in such a way that the ions can accumulate energy from the collisions, a situation which eventually leads to disintegration of the ions.
Although the ions can be produced in the interior, they can also be introduced from the outside. A collision gas in the ion trap ensures that the originally existing ion oscillations are decelerated and damped in the quadrupolar RF field; the ions then accumulate as a small cloud in the center of the ion trap. The diameter of the cloud in customary ion traps is usually about a millimeter; this is determined by an equilibrium between the centering effect of the RF field (the retroactive force of the pseudopotential) and the Coulomb forces of repulsion between the ions. The internal dimensions of commercial ion traps are usually characterized by a spacing between the end caps of about 14 mm, while the ring diameter is between 14 and 20 mm.
A common method for ionizing larger biomolecules is the electrospray procedure (ESI=electrospray ionization) whereby ions are ionized at atmospheric pressure outside the mass spectrometer. These ions are then introduced via well known admission systems into the vacuum of the mass spectrometer and from there into the ion trap.
Such ionization produces virtually no fragment ions; the ions are primarily those of the sprayed molecules. With electrospraying, however, multiply charged molecular ions are produced in large numbers. Due to the almost complete absence of fragment ions during the ionization process, the only information which can be acquired from the mass spectrum is the molecular weight of the molecule; no information is acquired regarding internal molecular structures, which might otherwise be used for further identifying the substance present. Such information can only be acquired when fragment ion spectra are recorded.
Recently, a procedure for fragmenting biomolecules, mainly peptides and proteins, has become known from Ion Cyclotron Resonance (ICR) or Fourier Transform Mass Spectrometry (FTMS). This involves allowing ions to capture low energy electrons, whereby the released ionization energy leads to the fracturing of usually chained molecules. The procedure has been termed ECD (electron capture dissociation). If the molecules are double charged, one of the two fragments stays in place as an ion. The fragmentation follows very simple rules (for experts: there are essentially only c breaks, and only very few y breaks of the amino acids of a peptide), so that the composition of the molecule can be deduced very easily from the fragmentation pattern. The sequence of peptides and proteins in particular can be easily seen from the fragmentation pattern. The interpretation of these ECD fragment spectra is less complicated than the interpretation of collisionally generated fragment spectra.
Although it is also possible to fragment singly or triply charged ions in this way, this procedure displays its best performance with doubly charged molecules. If an electrospray ionization is applied to peptides, the most frequently produced ions are usually doubly charged. Electrospray ionization is a method of ionization which is applied particularly often to biomolecules for mass spectroscopic studies in ion traps.
For fragmentation by electron capture, the kinetic energy of the electrons must be very low since no capture can occur otherwise. In practice, electrons are provided with an energy which is only marginally greater than the thermal energy of the electrons. This can be done extremely well in the very strong magnetic field of the Fourier transform mass spectrometer, since the electrons simply drift along the magnetic field lines until they reach the ion cloud.
However, in Paul electric RF ion traps this can not occur. As a rule, ion traps possess perforations in the end cap through which the ions can enter and leave. When ionization occurs internally the ionizing radiation is also introduced through this end cap perforation. For this purpose one usually uses an electron beam. The strongly oscillating RF field in the interior of the ion trap either accelerates the electrons so that they rush through the trap volume with considerable energy, or it repels the electrons already at the admission hole. Such electrons are hardly suited for electron capture. Only for an extremely short period of time, for fractions of nanoseconds during the periods when the RF voltage traverses zero, is there no field and can low energy electrons reach the ion cloud in a low energy form. However, this small number of low energy electrons coexists with many more electrons which have been accelerated to substantial energies; fragmentation by high-energy electron collision completely blankets fragmentation by electron capture and in this way renders the fragment ion spectra unusable.
In its simplest implementation, the procedure of the invention injects electrons into the ion trap not through one of the end cap perforations, but instead through an additionally made aperture in the ring electrode, while the electron source is kept at such a high positive potential that the oscillating potential at the center of the ion trap is only just achieved or exceeded (i.e. at the RF voltage maximum) for a very short period of a few nanoseconds. Only during this period can the electrons reach the ion cloud, decelerated to near zero kinetic energy, and thereby ideal for ion capture. At all other time-points the electrons are not capable of reaching the center of the ion trap since the potential of the center is more negative than that of the electron source so that it repels the always negatively charged electrons.
Deceleration of the electrons occurs on the way from the ring electrode to the center; the electrons must scale the saddle-like potential peak (see FIGS. 1 and 2). The ion cloud is located at the saddle point. In the z direction, i. e. the direction through both end caps, the saddle potential focuses the electrons on the ion cloud, and laterally deviating electrons are driven back to the correct course in the saddle well. In the r direction across the ring electrode, however, there acts a defocusing field, and only ions with the correct original direction can reach the ion cloud.
The low energy electrons are easily captured, in a first step, by the ion cloud (not yet by individual ions). Within the ion cloud, there exists a potential well capable to hold back the electrons. The capturing process is initiated by deflecting electrons in near hits with positive ions, thereby straying and capturing the electrons in the potential well. The electrons can be kept captured in the potential well even during the next cycles of the trap RF. This keeps the electrons ready for the next capturing step: capture of the electrons by the individual ions, leading to dissociation.
Fragmentation in the ion trap usually is performed at an RF voltage which is between a tenth and a fifth of the maximal voltage required for spectral recording. An RF voltage of e.g. around 3 kV (peak-to-peak) is set for fragmentation, and this voltage fluctuates sinusoidally in a range from xe2x88x921.5 to +1.5 kV (chassis or ground potential) at the ring. The end cap electrodes are held at ground potential. The center of the ion trap follows the ring potential so that it is always about half the ring electrode potential when the internal radius of the ring electrode is 1.4xc3x97 greater than the distance between the end cap electrodes, i.e. between xe2x88x92750 and +750 V. If the electron source is kept at a DC potential of +750 V, the electrons can reach the center only when the ring potential has a maximum potential of +1.5 kV so that the center has a potential +750 V. The electrons in this case are accelerated outside the ion trap from the potential of the electron source (+750 V) to the potential of the ring electrode (+1.5 kV) so that they gain an energy of 750 electron-volts. In the interior of the ion trap the kinetic energy of 750 eV is decelerated practically to 0 eV since a potential of +750 V prevails at the center. At all other time-points the center possesses a negative potential which repels the negatively charged electrons.
For an ion trap in which the distance between the end caps and the radius of the ring electrodes are more or less equal, the potential at the center is about ⅔ that of the ring electrode.
The conditions for allowing access of low energy electrons prevail only for a short period when the maximum RF voltage is present in the saddle. The duration is only approximately 1% of the oscillation cycle, i.e. approx. 10 nanoseconds. With an electron source, electron currents of approx. 100 xcexcA can be very easily achieved, i.e. 6xc3x97106 electrons in 10 nanoseconds. For a satisfactory spectrum only approximately 103 ions should be present in the ion cloud, since otherwise a deterioration of mass resolution occurs due to the effects of space charging. Even if a ten-fold greater number of ions is stored in order to compensate for losses in fragmentation yield, the number of electrons in a single high frequency period is still many times greater than the number of ions present. Since the supply of electrons can be maintained for a thousand or more high frequency cycles, a sufficiently large supply of electrons can easily be provided, even taking into account the defocusing effect in r direction.
The procedure can also be implemented involving injection of electrons through an aperture in one of the end caps. In this case, however, the end caps should be supplied with RF voltage so that they are both in-phase (commercial ion traps usually do not offer this option), and the ring electrode should be held at the chassis potential. The invention also embraces an ion trap mass spectrometer for implementing the procedure, with at least one aperture in the ring electrode and with an electron source whose electron generation potential can be adjusted to the required voltage.